Methods of using 3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione for the treatment and management of myeloproliferative diseases

ABSTRACT

Methods of treating, preventing and/or managing a myeloproliferative disease are disclosed. Specific methods encompass the administration of an immunomodulatory compound, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, alone or in combination with a second active agent, and/or the transplantation of blood or cells. Particular second active agents are capable of suppressing the overproduction of hematopoietic stem cells or ameliorating one or more of the symptoms of a myeloproliferative disease. Pharmaceutical compositions, single unit dosage forms, and kits suitable for use in methods of the invention are also disclosed.

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/424,730 filed on Nov. 6, 2002, the entirety of which isincorporated herein by reference.

1. FIELD OF THE INVENTION

This invention relates to methods of treating, preventing and/ormanaging myeloproliferative diseases and related syndromes whichcomprise the administration of immunomodulatory compounds alone or incombination with other therapies.

2. BACKGROUND OF THE INVENTION

2.1 Pathobiology of MPD

Myeloproliferative disease (MPD) refers to a group of disorderscharacterized by clonal abnormalities of the hematopoictic stem cell.See e.g., Current Medical Diagnosis & Treatment, pp. 499 (37^(th) ed.,Tierney et al. ed, Appleton & Lange, 1998). Since the stem cell givesrise to myeloid, erythroid, and platelet cells, qualitative andquantitative changes can be seen in all these cell lines. Id.

MPD is further subdivided on the basis of the predominantlyproliferating myeloid cell type. Erythrocyte excess is classified as“polycythemia rubra vera (PRV)” or “polycythemia vera,” platelet excessas “primary (or essential) thromobocythemia (PT),” and granulocyteexcess as “chronic myelogenous leukemia (CML).” A fourth subcategory ofMPD is “agnogenic myeloid metaplasia (AMM),” which is characterized bybone marrow fibrosis and extramedullary hematopoiesis. Cecil Textbook ofMedicine, pp. 922 (20^(th) ed., Bennett and Plum ed., W.B. SaundersCompany, 1996). These disorders are grouped together because the diseasemay evolve from one form into another and because hybrid disorders arecommonly seen. Tierney et al, supra, at pp. 499. All of themyeloproliferative disorders may progress to acute leukemia naturally oras a consequence of mutagenic treatment. Id.

Most patients with PRV present symptoms related to expanded blood volumeand increased blood viscosity. Id. at pp. 500. Common complaints includeheadache, dizziness, tinnitus, blurred vision, and fatigue. Id. Thespleen is palpably enlarged in 75% of cases, but splenomegaly is nearlyalways present when imaged. Id. Thrombosis is the most commoncomplication of PRV and the major cause of morbidity and death in thisdisorder. Thrombosis appears to be related to increased blood viscosityand abnormal platelet function. Id. Sixty percent of patients with PRVare male, and the median age at presentation is 60. It rarely occurs inadults under age 40. Id.

Thrombosis is also a common complication in patients suffering from PT.Cecil Textbook of Medicine, pp. 922 (20^(th) ed., Bennett and Plum ed.,W.B. Saunders Company, 1996). A platelet count ≧6×10⁵ per microliter hasbeen set to diagnose PT. Tefferi et al., Mayo Clin Proc 69:651 (1994).Most patients are asymptomatic when PT is diagnosed, usually throughincidental discovery of increased peripheral blood platelet count.Bennett and Plum, supra, at pp. 922. Approximately one quarter, however,have either thrombotic or hemorrhagic events. Id. PT rarely transformsinto acute leukemia or AMM, and most patients have a normal lifeexpectancy. Id. at pp. 923. However, at least one third of patients withPT eventually undergo major thrombohemorrhage complications. Id.

AMM is characterized by fibrosis of the bone marrow, splenomegaly, and aleukoerythroblastic peripheral blood picture with teardroppoikilocytosis. Id. at pp. 502. AMM develops in adults over age 50 andis usually insidious in onset. Id. Later in the course of the disease,bone marrow failure takes place as the marrow becomes progressively morefibrotic. Id. Anemia becomes severe. Id. Painful episodes of splenicinfarction may occur. Severe bone pain and liver failure also occur inthe late stage of AMM. Id. The median survival from time of diagnosis isapproximately 5 years. Id. at pp. 503.

The precise cause of MPD is not clear. Current data suggest some growthfactors are involved. For instance, in both PRV and PT, in contrast tonormal erythroid progenitor cells, polycythemia vera erythroidprogenitor cells can grow in vitro in the absence of erythropoietin dueto hypersensitivity to insulin like growth factor I. Harrison'sPrinciples of Internal Medicine, pp. 701 (15^(th) ed., Braunwald et al.ed., McGraw-Hill, 2001). In AMM, the overproduction of type III collagenhas been attributed to platelet-derived growth factor or transforminggrowth factor β (TGF-β). Id. at pp. 703; see also, Martyr, Leuk Lymphoma6:1 (1991).

In some MPD forms, specific chromosomal changes are seen. For instance,nonrandom chromosome abnormalities, such as 20q-, trisomy 8 or 9 havebeen documented in a small percentage of untreated PRV patients, and20q-, 13q-, trisomy 1q are common in AMM patient. Harrison's Principlesof Internal Medicine, pp. 701-3 (15^(th) ed., Braunwald et al. ed.,McGraw-Hill, 2001). Philadelphia chromosome is present in the bonemarrow cells of more than 90% of patients with typical CML and somepatients with PRV. See e.g., Kurzrock et al., N Engl J Med 319:990(1988). The Philadelphia chromosome results from a balancedtranslocation of material between the long arms of chromosomes 9 and 22.The break, which occurs at band q34 of the long arm of chromosome 9,allows translocation of the cellular oncogene C-ABL to a position onchromosome 22 called the breakpoint cluster region (bcr). The appositionof these two genetic sequences produces a new hybrid gene (BCR/ABL),which codes for a novel protein of molecular weight 210,000 kD (P210).The P210 protein, a tyrosine kinase, may play a role in triggering theuncontrolled proliferation of CML cells. See e.g., Daley et al., Science247:824 (1990).

The incidence of MPD varies depending on the form of the disease. PRV isdiagnosed in 5-17 persons per 1,000,000 per year. Cecil Textbook ofMedicine, pp. 920-926 (20^(th) ed., Bennett and Plum ed., W.B. SaundersCompany, 1996). True incidences of PT and AMM are not known becauseepidemiological studies on these disorders are inadequate. Id.Internationally, PRV is reportedly lower in Japan, i.e., 2 person per1,000,000 per year. Id.

2.2 MPD Treatment

The treatment of choice for PRV is phlebotomy. Current Medical Diagnosis& Treatment, pp. 501 (37^(th) ed., Tierney et al. ed, Appleton & Lange,1998). One unit of blood (approximately 500 mL) is removed weekly untilthe hematocrit is less than 45%. Id. Because repeated phlebotomyproduces iron deficiency, the requirement for phlebotomy has to begradually decreased. Id. It is important to avoid medicinal ironsupplementation, as this can thwart the goals of a phlebotomy program.Id.

In more severe cases of PRV, myelosuppressive therapy is used. Id. Oneof the widely used myelosuppressive agents is hydroxyurea. Id.Hydroxyurea is an oral agent that inhibits ribonucleotide reductase.Bennett and Plum, supra, at pp. 924. The usual dose is 500-1500 mg/dorally, adjusted to keep platelets <500,000/μL without reducing theneurophil count to <2000/μL. Tierney et al., supra, at pp. 501. Sideeffects of hydroxyurea include mild gastrointestinal complaints,reversible neutropenia, and mucocutaneous lesions. Bennett and Plum,supra, at pp. 924. Busulfan may also be used in a dose of 4-6 mg/d for4-8 weeks. Tierney et al, supra, at pp. 501. Alpha interferon has beenshown to have some ability to control the disease. The usual dose is 2-5million units subcutaneously three times weekly. Id. Anagrelide has alsobeen approved for use in treatment of thrombocytosis. Id. Some of themyelosuppressive agents, such as alkylating agents and radiophosphorus(³²P), have been shown to increase the risk of conversion of PRV toacute leukemia. Id. Using myelosuppressive agents for long period maycause prolonged severe myelosuppression.

Most authorities agree that treatment of PT should be aimed atdecreasing the level of platelets in patients with a history ofthrombosis as well as those with cardiovascular risk factors. Bennettand Plum, supra, at pp. 923. However, the benefit of specific therapyhas not been established, and there is concern about the leukemogenicpotential of the available therapeutic agents. Id. When treatment isdecided upon, the initial drugs are hydroxyurea or anagrelide. Id. atpp. 924. Anagrelide is an oral agent that may involve inhibition ofmegakaryocyte maturation. Id. The starting dose is 0.5 mg given fourtimes a day. Id. It is relatively contraindicated in elderly patientswith heart disease. Id. Alpha interferon can also be used in thetreatment of PT. Id.

Currently, there is no specific treatment for AMM. Tierney et al.,supra, at pp. 502. The management of AMM is directed to symptoms. Anemicpatients are supported with red blood cells in transfusion. Id.Androgens such as oxymetholone, 200 mg orally daily, or testosteronehelp reduce the transfusion requirement in one third of cases but arepoorly tolerated by women. Id. Splenectomy is indicated for splenicenlargement that causes recurrent painful episodes, severethrombocytopenia, or an unacceptable high red blood cell transfusionrequirement. Id. Alpha interferon (2-5 million units subcutaneouslythree times weekly) leads to improvement in some cases. Id.

Since most therapies used in the treatment of MPD are targeted only atsymptoms, and most agents used have serious side effects, with thedanger of causing severe myelosuppression or converting the disorder toacute leukemia, there is a great need to find new treatments of MPD thateither target the underlying cause of the disorder or improve theeffectiveness and safety of the current treatments.

2.3 Thalidomide and other Compounds Useful in the Treatment of Disease

Thalidomide is a racemic compound sold under the tradename Thalomid® andchemically named α-(N-phthalimido)glutarimide or2-(2,6-dioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione. Thalidomide wasoriginally developed in the 1950's to treat morning sickness, but due toits teratogenic effects was withdrawn from use. Thalidomide has beenapproved in the United States for the acute treatment of the cutaneousmanifestations of erythema nodosum leprosum in leprosy. Physicians' DeskReference, 1154-1158 (56^(th) ed., 2002). Because its administration topregnant women can cause birth defects, the sale of thalidomide isstrictly controlled. Id. Thalidomide has reportedly been studied in thetreatment of other diseases, such as chronic graft-vs-host disease,rheumatoid arthritis, sarcoidosis, several inflammatory skin diseases,and inflammatory bowel disease. See generally, Koch, H. P., Prog. Med.Chem. 22:165-242 (1985). See also, Moller, D. R., et al., J. Immunol.159:5157-5161 (1997); Vasiliauskas, E. A., et al., Gastroenterology117:1278-1287 (1999); Ehrenpreis, E. D., et al., Gastroenterology117:1271-1277 (1999). It has further been alleged that thalidomide canbe combined with other drugs to treat ischemia/repercussion associatedwith coronary and cerebral occlusion. See U.S. Pat. No. 5,643,915, whichis incorporated herein by reference.

More recently, thalidomide was found to exert immunomodulatory andanti-inflammatory effects in a variety of disease states, cachexia inAIDS, and opportunic infections in AIDS. In studies to define thephysiological targets of thalidomide, the drug was found to have a widevariety of biological activities exclusive of its sedative effectincluding neurotoxicity, teratogenicity, suppression of TNF-α productionby monocytes/macrophages and the accompanying inflammatory toxicitiesassociated with high levels of TNF-α, and inhibition of angiogenesis andneovascularization.

Additionally, beneficial effects have been observed in a variety ofdermatological conditions, ulcerative colitis, Crohn's disease,Bechets's syndrome, systemic lupus erythematosis, aphthous ulcers, andlupus. The anti-angiogenic properties of thalidomide in in vivo modelshave been reported. D'Amato et al., Thalidomide Is An Inhibitor OfAngiogenesis, 1994, PNAS, USA 91:4082-4085.

One of the most therapeutically significant potential uses ofthalidomide is in the treatment of cancer. The compound has beeninvestigated in the treatment of various types of cancer, such asrefractory multiple myeloma, brain, breast, colon, and prostate cancer,melanoma, mesothelioma, and renal cell carcinoma. See, e.g., Singhal,S., et al., New England J. Med. 341(21):1565-1571 (1999); and Marx, G.M., et al, Proc. Am. Soc. Clin. Oncology 18:454a (1999). Thalidomidereportedly can also be used to prevent the development of chroniccardiomyopathy in rats caused by doxorubicin. Costa, P. T., et al.,Blood 92(10:suppl. 1):235b (1998). Other reports concerning the use ofthalidomide in the treatment of specific cancers include its combinationwith carboplatin in the treatment of glioblastoma multiforme. McCann,J., Drug Topics 41-42 (Jun. 21, 1999). The use of thalidomide incombination with dexamethasone reportedly was effective in the treatmentof patients suffering from multiple myeloma who also received, assupportive care, human granulocyte colony-stimulating factor (G-CSF),ciprofloxacin, and non-absorbable antifungal agents. Kropff, M. H.,Blood 96(11 part 1):168a (2000); see also, Munshi, N. et al., Blood94(10 part 1):578a (1999). Other chemotherapy combinations that comprisethalidomide are disclosed in International Application No.PCT/US01/15326 to R. Govindarjan and A. Zeitlan, and in InternationalApplication No. PCT/US01/15327 to J. B. Zeldis, et al.

In an effort to provide compounds that have greater therapeutic safetyand efficacy than thalidomide, researchers have begun investigating alarge number of other compounds, some of which are derivatives ofthalidomide. See, e.g., Marriott, J. B., et al., Expert Opin. Biol.Ther. 1(4):1-8 (2001); G. W. Muller, et al., Journal of MedicinalChemistry 39(17): 3238-3240 (1996); and G. W. Muller, et al., Bioorganic& Medicinal Chemistry Letters 8: 2669-2674 (1998). Examples include, butare not limited to, the substituted 2-(2,6-dioxopiperidin-3-yl)phthalimies and substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolesdescribed in U.S. Pat. Nos. 6,281,230 and 6,316,471, both to G. W.Muller, et al.

A group of compounds selected for their capacity to potently inhibitTNF-α production by LPS stimulated PBMC has been investigated. L. G.Corral, et al., Ann. Rheum. Dis. 58:(Suppl I) 1107-1113 (1999). Thesecompounds, which are referred to as IMiDs™ or Immunomodulatory Drugs,show not only potent inhibition of TNF-α but also marked inhibition ofLPS induced monocyte IL1β and IL12 production. LPS induced IL6 is alsoinhibited by IMiDs™, albeit partially. These compounds are potentstimulators of LPS induced IL10, increasing IL10 levels by 200 to 300%.Id.

While many such compounds have shown promise as therapeutic agents,their mechanisms of action and effectiveness are still underinvestigation. Moreover, there remains a need for therapeutic agents totreat MPD and its related disorders.

3. SUMMARY OF THE INVENTION

This invention encompasses methods of treating and preventingmyeloproliferative disease (“MPD”) which comprise administering to apatient in need thereof a therapeutically or prophylactically effectiveamount of an immunomodulatory compound of the invention, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof. The invention also encompasses methods ofmanaging MPD (e.g., lengthening the time of remission) which compriseadministering to a patient in need of such management a therapeuticallyor prophylactically effective amount of an immunomodulatory compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stercoisomer, clathrate, or prodrug thereof.

One embodiment of the invention encompasses the use of one or moreimmunomodulatory compounds in combination with conventional therapiespresently used to treat, prevent or manage MPD such as, but not limitedto, hydroxyurea, anagrelide, interferons, kinase inhibitors, cancerchemotherapeutics, stem cell transplanation and other transplantations.

Another embodiment of the invention encompasses a method of reducing orpreventing an adverse effect associated with MPD therapy, whichcomprises administering to a patient in need of such treatment orprevention an amount of an immunomodulatory compound of the invention,or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof, that is sufficient to reduce an adverseeffect associated with the MPD therapy. This emodiment includes the useof an immunomodulatory compound of the invention to protect against ortreat an adverse effect associated with the use of the MPD therapy. Thisembodiment encompasses raising a patient's tolerance for the MPDtherapy.

Another embodiment of the invention encompasses a method of increasingthe therapeutic efficacy of a MPD treatment which comprisesadministering to a patient in need of such increased therapeuticefficacy an amount of an immunomodulatory compound of the invention, ora pharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof, that is sufficient to increase thetherapeutic efficacy of the MPD treatment.

The invention further encompasses pharmaceutical compositions, singleunit dosage forms, and kits suitable for use in treating, preventingand/or managing MPD, which comprise an immunomodulatory compound of theinvention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof.

4. DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the invention encompasses methods of treating orpreventing MPD, which comprise administering to a patient in need ofsuch treatment or prevention a therapeutically or prophylacticallyeffective amount of an immunomodulatory compound or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof. The embodiment encompasses the treatment, prevention ormanagement of specific sub-types of MPD such as, but not limited to,polycythemia rubra vera (PRV), primary thromobocythemia (PT), andagnogenic myeloid metaplasia (AMM).

As used herein, the term “myeloproliferative disease,” or “MPD,” means ahematopoietic stem cell disorder characterized by one or more of thefollowing: tonal expansion of a multipotent hematopoietic progenitorcell with the overproduction of one or more of the formed elements ofthe blood (e.g., elevated red blood cell count, elevated white bloodcell count, and/or elevated platelet count), presence of Philadelphiachromosome or bcr-abl gene, teardrop poikilocytosis on peripheral bloodsmear, leukoerythroblastic blood pictuer, giant abnormal platelets,hypercellular bone marrow with reticular or collagen fibrosis, markedleft-shifted myeloid series with a low percentage of promyclocytes andblasts, splenomegaly, thrombosis, risk of progression to acute leukemiaor cellular marrow with impaired morphology. The term“myeloproliferative disease,” or “MPD,” unless otherwise noted includes:polycythemia rubra vera (PRV), primary thromobocythemia (PT), andagnogenic myeloid metaplasia (AMM). In a specific embodiment, the term“myeloproliferative disease” or “MPD” excludes leukemia. Particulartypes of MPD are PRV, PT, and AMM.

Another embodiment of the invention encompasses methods of managing MPDwhich comprises administering to a patient in need of such management aprophylactically effective amount of an immunomodulatory compound or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof.

Another embodiment of the invention encompasses a pharmaceuticalcomposition comprising an immunomodulatory compound or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof.

Also encompassed by the invention are single unit dosage formscomprising an immunomodulatory compound or a pharmaceutically acceptablesalt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof.

Another embodiment of the invention encompasses a method of treating,preventing and/or managing MPD, which comprises administering to apatient in need of such treatment, prevention and/or management atherapeutically or prophylactically effective amount of animmunomodulatory compound or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, and atherapeutically or prophylactically effective amount of a second activeagent.

Examples of second active agents include, but are not limited to,cytokines, corticosteroids, ribonucleotide reductase inhibitors,platelet inhibitors, all-trans retinoic acids, kinase inhibitors,topoisomerase inhibitors, farnesyl transferase inhibitors, antisenseoligonucleotides, vaccines, anti-cancer agents, anti-fungal agents,anti-inflammatory agents, immunosuppressive or myelosuppressive agents,and conventional therapies for MPD.

Without being limited by theory, it is believed that certainimmunomodulatory compounds of the invention can act in complementary orsynergistic ways with conventional and other therapies in the treatmentor management of MPD. It is also believed that certain immunomodulatorycompounds of the invention act by different mechanisms than conventionaland other therapies in the treatment or management of MPD. In addition,it is believed that certain immunomodulatory compounds of the inventionare effective when administered to patients who are refractory toconventional treatments for myeloproliferative diseases as well astreatments using thalidomide. As used herein, the term “refractory”means the patient's response to a MPD treatment is not satisfactory byclinical standards, e.g., show no or little improvement of symptoms orlaboratory findings.

It is also believed that certain therapies may reduce or eliminateparticular adverse effects associated with some immunomodulatorycompounds, thereby allowing the administration of larger amounts of animmunomodulatory compound to patients and/or increasing patientcompliance. It is further believed that some immunomodulatory compoundsmay reduce or eliminate particular adverse effects associated with otherMPD therapies, thereby allowing the administration of larger amounts ofsuch therapies to patients and/or increasing patient compliance.

Another embodiment of the invention encompasses a kit comprising: apharmaceutical composition comprising an immunomodulatory compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof and a second active agentand/or instructions for use. The invention further encompasses kitscomprising single unit dosage forms.

Another embodiment of the invention encompasses a method of reversing,reducing or avoiding an adverse effect associated with theadministration of an active agent used to treat MPD in a patientsuffering from MPD, which comprises administering to a patient in needthereof a therapeutically or prophylactically effective amount of animmunomodulatory compound or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof. Examplesof active agents include, but are not limited to, the second activeagents described herein (see section 4.2.).

Examples of adverse effects associated with active agents used to treatMPD include, but are not limited to: conversion to acute leukemia;severe myelosuppression; gastrointestinal toxicity such as, but notlimited to, early and late-forming diarrhea and flatulence;gastrointestinal bleeding; nausea; vomiting; anorexia; leukopenia;anemia; neutropenia; asthenia; abdominal cramping; fever; pain; loss ofbody weight; dehydration; alopecia; dyspnea; insomnia; dizziness,mucositis, xerostomia, mucocutaneous lesions, and kidney failure.

As leukemic transformation develops in certain stages of MPD,transplantation of peripheral blood stem cells, hematopoietic stem cellpreparation or bone marrow may be necessary. Without being limited bytheory, it is believed that the combined use of an immunomodulatorycompound and the transplantation of stem cells in a patient sufferingfrom MPD provides a unique and unexpected synergism. In particular, itis believed that certain immunomodulatory compounds of the inventionexhibit immunomodulatory activity that can provide additive orsynergistic effects when given concurrently with transplantationtherapy. Immunomodulatory compounds of the invention can work incombination with transplantation therapy to reduce complicationsassociated with the invasive procedure of transplantation and risk ofrelated Graft Versus Host Disease (GVHD). Therefore, this inventionencompasses a method of treating, preventing and/or managing MPD, whichcomprises administering to a patient (e.g., a human) an immunomodulatorycompound of the invention, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, before,during, or after transplantation therapy.

The invention also encompasses pharmaceutical compositions, single unitdosage forms, and kits which comprise one or more immunomodulatorycompounds or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof, a second active ingredient,and/or blood or cells for transplantation therapy. For example, a kitmay contain one or more compounds of the invention, stem cells fortransplantation and an immunosuppressive agent, and an antibiotic orother drug.

4.1 Immunomodulatory Compounds

Compounds used in the invention include immunomodulatory compounds thatare racemic, stereomerically enriched or stereomerically pure, andpharmaceutically acceptable salts, solvates, hydrates, stereoisomers,clathrates, and prodrugs thereof. Preferred compounds used in theinvention are small organic molecules having a molecular weight lessthan about 1000 g/mol, and are not proteins, peptides, oligonucleotides,oligosaccharides or other macromolecules.

As used herein and unless otherwise indicated, the term “stereomericallypure” means a composition that comprises one stereoisomer of a compoundand is substantially free of other stercoisomers of that compound. Forexample, a stereomerically pure composition of a compound having onechiral center will be substantially free of the opposite enantiomer ofthe compound. A stereomerically pure composition of a compound havingtwo chiral centers will be substantially free of other diastereomers ofthe compound. A typical stereomerically pure compound comprises greaterthan about 80% by weight of one stereoisomer of the compound and lessthan about 20% by weight of other stereoisomers of the compound, morepreferably greater than about 90% by weight of one stereoisomer of thecompound and less than about 10% by weight of the other stereoisomers ofthe compound, even more preferably greater than about 95% by weight ofone stereoisomer of the compound and less than about 5% by weight of theother stereoisomers of the compound, and most preferably greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound. As usedherein and unless otherwise indicated, the term “stereomericallyenriched” means a composition that comprises greater than about 60% byweight of one stereoisomer of a compound, preferably greater than about70% by weight, more preferably greater than about 80% by weight of onestereoisomer of a compound. As used herein and unless otherwiseindicated, the term “enantiomerically pure” means a stereomerically purecomposition of a compound having one chiral center. Similarly, the term“stereomerically enriched” means a stereomerically enriched compositionof a compound having one chiral center.

As used herein and unless otherwise indicated, the term“immunomodulatory compounds” or “IMiDs™” (Celgene Corporation) usedherein encompasses small organic molecules that markedly inhibit TNF-α,LPS induced monocyte IL1β and IL12, and partially inhibit IL6production. Specific immunomodulatory compounds of the invention arediscussed below.

TNF-α is an inflammatory cytokine produced by macrophages and monocytesduring acute inflammation. TNF-α is responsible for a diverse range ofsignaling events within cells. TNF-α may play a pathological role incancer. Without being limited by particular theory, one of thebiological effects exerted by the immunomodulatory compounds of theinvention is the reduction of synthesis of TNF-α. Immunomodulatorycompounds of the invention enhance the degradation of TNF-α mRNA.

Further, without being limited by particular theory, immunomodulatorycompounds used in the invention may also be potent co-stimulators of Tcells and increase cell proliferation dramatically in a dose dependentmanner. Immunomodulatory compounds of the invention may also have agreater co-stimulatory effect on the CD8+ T cell subset than on the CD4+T cell subset. In addition, the compounds preferably haveanti-inflammatory properties, and efficiently co-stimulate T cells.

Specific examples of immunomodulatory compounds of the invention,include, but are not limited to, cyano and carboxy derivatives ofsubstituted styrenes such as those disclosed in U.S. Pat. No. 5,929,117;1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl) isoindolines and1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such asthose described in U.S. Pat. No. 5,874,448; the tetra substituted2-(2,6-dioxopiperdin-3-yl)-1-oxoisoindolines described in U.S. Pat. No.5,798,368; 1-oxo and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines(e.g., 4-methyl derivatives of thalidomide and EM-12), including, butnot limited to, those disclosed in U.S. Pat. No. 5,635,517; and a classof non-polypeptide cyclic amides disclosed in U.S. Pat. Nos. 5,698,579and 5,877,200; analogs and derivatives of thalidomide, includinghydrolysis products, metabolites, derivatives and precursors ofthalidomide, such as those described in U.S. Pat. Nos. 5,593,990,5,629,327, and 6,071,948 to D'Amato; aminothalidomide, as well asanalogs, hydrolysis products, metabolites, derivatives and precursors ofaminothalidomide, and substituted 2-(2,6-dioxopiperidin-3-yl)phthalimides and substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolessuch as those described in U.S. Pat. Nos. 6,281,230 and 6,316,471;isoindole-imide compounds such as those described in U.S. patentapplication Ser. No. 09/972,487 filed on Oct. 5, 2001, U.S. patentapplication Ser. No. 10/032,286 filed on Dec. 21, 2001, andInternational Application No. PCT/US01/50401 (International PublicationNo. WO 02/059106). The entireties of each of the patents identifiedherein are incorporated herein by reference. Immunomodulatory compoundsof the invention do not include thalidomide.

Other specific immunomodulatory compounds of the invention include, butare not limited to, 1-oxo-and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolines substituted with amino in the benzo ring as described inU.S. Pat. No. 5,635,517 which is incorporated herein. These compoundshave the structure I:

in which one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, in particular methyl. Specificimmunomodulatory compounds include, but are not limited to:

1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;

1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline;

1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-aminoisoindoline;

1-oxo-2-(2,6-dioxopiperidin-3-yl)-7-aminoisoindoline;

1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;

and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline.

Other specific immunomodulatory compounds of the invention belong to aclass of substituted 2-(2,6-dioxopiperidin-3-yl) phthalimides andsubstituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles, such as thosedescribed in U.S. Pat. Nos. 6,281,230; 6,316,471; 6,335,349; and6,476,052, and International Patent Application No. PCT/US97/13375(International Publication No. WO 98/03502), each of which isincorporated herein. Compounds representative of this class are of theformulas:

wherein R¹ is hydrogen or methyl. In a separate embodiment, theinvention encompasses the use of enantiomerically pure forms (e.g.optically pure (R) or (S) enantiomers) of these compounds.

Still other specific immunomodulatory compounds of the invention belongto a class of isoindole-imides disclosed in U.S. patent application Ser.Nos. 10/032,286 and 09/972,487, and International Application No.PCT/US01/50401(International Publication No. WO 02/059106), each ofwhich are incorporated herein by reference. Representative compounds areof formula II:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-(C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂-C₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl benzyl, aryl, (C₂-C₅)heteroaryl, or (C₀-C₈)alkyl-C(O)O—R⁵or the R⁶ groups can join to form a heterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center.

In specific compounds of formula II, when n is 0 then R1 is(C3-C7)cycloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, benzyl, aryl,(C0-C4)alkyl-(C1-C6)heterocycloalkyl, (C0-C4)alkyl-(C2-C5)heteroaryl,C(O)R3, C(O)OR4, (C1-C8)alkyl-N(R6)2, (C1-C8)alkyl-OR5,(C1-C8)alkyl-C(O)OR5, C(S)NHR3, or (C1-C8)alkyl-O(CO)R5;

R² is H or (C₁-C₈)alkyl; and

R³ is (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₅-C₈)alkyl-N(R⁶)₂;(C₀-C₈)alkyl-NH—C(O)O—R⁵; (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵; and the other variables have the samedefinitions.

In other specific compounds of formula II, R² is H or (C₁-C₄)alkyl.

In other specific compounds of formula II, R¹ is (C₁-C₈)alkyl or benzyl.

In other specific compounds of formula II, R¹ is H, (C₁-C₈)alkyl,benzyl, CH₂OCHH₃, CH₂CH₂OCHH₃, or

In another embodiment of the compounds of formula II, R¹ is

wherein Q is O or S, and each occurrence of R⁷ is independently H,(C₁-C₈)alkyl, benzyl, CH₂OCHH₃, or CH₂CH₂OCHH₃.

In other specific compounds of formula II, R¹ is C(O)R³.

In other specific compounds of formula II, R³ is(C₀-C₄)alkyl-(C₂-C₄)heteroaryl, (C₁-C₈)alkyl, aryl, or (C₀-C₄)alkyl-OR⁵.

In other specific compounds of formula II, heteroaryl is pyridyl, furyl,or thienyl.

In other specific compounds of formula II, R¹ is C(O)OR⁴.

In other specific compounds of formula II, the H of C(O)NHC(O) can bereplaced with (C₁-C₄)alkyl, aryl, or benzyl.

Still other specific immunomodulatory compounds of the invention belongto a class of isoindole-imides disclosed in U.S. patent application Ser.No. 09/781,179, International Publication No. WO 98/54170, and U.S. Pat.No. 6,395,754, each of which are incorporated herein by reference.Representative compounds are of formula III:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH2OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n) H_(2n))— in which n has avalue of 0 to 4;

each of R8 and R9 taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R8 and R9 taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂[X]X₁CH₂CH₂— in which [X]X₁ is—O—, —S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center.

The most preferred immunomodulatory compounds of the invention are4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Thecompounds can be obtained via standard, synthetic methods (see e.g.,U.S. Pat. No. 5,635,517, incorporated herein by reference). Thecompounds are available from Celgene Corporation, Warren, N.J.4-(Amino)-2-(2,6-dioxo (3-piperidyl))-isoindoline-1,3-dione(ACTIMID™)has the following chemical structure:

3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(REVIMID™) has the following chemical structure:

The compounds of the invention can either be commercially purchased orprepared according to the methods described in the patents or patentpublications disclosed herein. Further, optically pure compounds can beasymmetrically synthesized or resolved using known resolving agents orchiral columns as well as other standard synthetic organic chemistrytechniques.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” encompasses non-toxic acid and baseaddition salts of the compound to which the term refers. Acceptablenon-toxic acid addition salts include those derived from organic andinorganic acids or bases know in the art, which include, for example,hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinicacid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,salicylic acid, phthalic acid, embolic acid, enanthic acid, and thelike.

Compounds that are acidic in nature are capable of forming salts withvarious pharmaceutically acceptable bases. The bases that can be used toprepare pharmaceutically acceptable base addition salts of such acidiccompounds are those that form non-toxic base addition salts, i.e., saltscontaining pharmacologically acceptable cations such as, but not limitedto, alkali metal or alkaline earth metal salts and the calcium,magnesium, sodium or potassium salts in particular. Suitable organicbases include, but are not limited to, N,N-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine(N-methylglucamine), lysine, and procaine.

As used herein and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide thecompound. Examples of prodrugs include, but are not limited to,derivatives of immunomodulatory compounds of the invention that comprisebiohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzableesters, biohydrolyzable carbamates, biohydrolyzable carbonates,biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Otherexamples of prodrugs include derivatives of immunomodulatory compoundsof the invention that comprise —NO, —NO₂, —ONO, or —ONO₂ moieties.Prodrugs can typically be prepared using well-known methods, such asthose described in 1 Burger's Medicinal Chemistry and Drug Discovery,172-178, 949-982 (Manfred E. Wolff ed., 5th ed. 1995), and Design ofProdrugs (H. Bundgaard ed., Elselvier, N.Y. 1985).

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,”“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl,acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, andpivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such asmethoxycarbonyloxymethyl, ethoxycarbonyloxyethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters,and acylamino alkyl esters (such as acetamidomethyl esters). Examples ofbiohydrolyzable amides include, but are not limited to, lower alkylamides, α-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

It should be noted that if there is a discrepancy between a depictedstructure and a name given that structure, the depicted structure is tobe accorded more weight. In addition, if the stereochemistry of astructure or a portion of a structure is not indicated with, forexample, bold or dashed lines, the structure or portion of the structureis to be interpreted as encompassing all stereoisomers of it.

4.2 Second Active Agents

One or more active ingredients can be used in combination with animmunomodulatory compound of the present invention. Preferably, thesecond active ingredient, or agent, is capable of suppressing theoverproduction of hematopoietic stem cells, or ameliorating one or moreof the symptoms of MPD.

Second active agents can be, but are not limited to, small molecules(e.g., synthetic inorganic, organometallic, or organic molecules), largemolecules, synthetic drugs, peptides, polypeptides, proteins, nucleicacids, antibodies and the like. Any agent that is known to be useful, orthat has been used or is currently being used for the prevention,treatment or amelioration of one or more symptoms of MPD can be used inthe combination with the present invention. Particular agents include,but are not limited to, anticancer agents (e.g., antimetabolites,antibiotics, alkylating agents, microtubule inhibitors, steroidhormones, DNA-repair enzyme inhibitors, kinase inhibitors, famesyltransferase inhibitors, antisense oligonucleotides, immunomodulators,antibodies, vaccines, and adnosine deaminase inhibitors), all-transretinoic acid (e.g., arsenic trioxide), platelet inhibitors (e.g.,aspirin, dipyridamole, ticlopidine, anagrelide), anticoagulants (e.g.,enoxaprin, heparin, warfarin), thrombolytic agents (e.g., alteplase(tPA), anistreplase, streptokinase, urokinase), antifibrosis agents(e.g., penicillamine, suramin, clochicine), agents used in treatingbleeding (e.g., aminocaproic acid, protamine sulfate, vitamin K), andagents used in treating anemia (e.g., vitamin K, folic acid).

This invention also encompasses the use of native, naturally occurring,and recombinant proteins. The invention further encompasses mutants andderivatives (e.g., modified forms) of naturally occurring proteins thatexhibit, in vivo, at least some of the pharmacological activity of theproteins upon which they are based. Examples of mutants include, but arenot limited to, proteins that have one or more amino acid residues thatdiffer from the corresponding residues in the naturally occurring formsof the proteins. Also encompassed by the term “mutants” are proteinsthat lack carbohydrate moieties normally present in their naturallyoccurring forms (e.g., nonglycosylated forms). Examples of derivativesinclude, but are not limited to, pegylated derivatives and fusionproteins, such as proteins formed by fusing IgG1 or IgG3 to the proteinor active portion of the protein of interest. See, e.g., Penichet, M. L.and Morrison, S. L., J. Immunol. Methods 248:91-101 (2001).

This invention further encompasses the use of immune cells ortransplantation of blood and marrow stem cells. For example, CMLpatients can be treated with infusion of donor white blood cells thatsuppress the growth of leukemia cells. Slavin et al., Transfus ApheresisSci 27(2):159-66 (2002).

Examples of anti-cancer drugs that can be used in the variousembodiments of the invention, including the methods, dosing regimens,cocktails, pharmaceutical compositions and dosage forms and kits of theinvention, include, but are not limited to: acivicin; aclarubicin;acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethan immunomodulatory compoundof the inventione; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib(COX-2 inhibitor); chlorambucil; cirolemycin; cisplatin; cladribine;crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine;dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin;dezaguanine; dezaguanine mesylate; diaziquone; dacarbazine; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; elfornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including-recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan; irinotecan hydrochloride; lanreotide acetate;letrozole; leuprolide acetate; liarozole hydrochloride; lometrexolsodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletan immunomodulatory compound of theinventione; safingol; safingol hydrochloride; semustine; simtrazene;sparfosate sodium; sparsomycin; spirogermanium hydrochloride;spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur;talisomycin; tecogalan sodium; taxotere; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermnine; cecropinB; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;elfornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; an immunomodulatory compound of the inventionazoacridones;imiquimod; immunostimulant peptides; insulin-like growth factor-1receptor inhibitor; interferon agonists; interferons; interleukins;iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemiainhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O⁶-benzylguanine; oblimersen; octreotide; okicenone; oligonucleotides;onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer;ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxelanalogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; rasinhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186etidronate; rhizoxin; ribozymes; RII retinamide; rogletanimmunomodulatory compound of the inventione; rohitukine; romurtide;roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU;sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescencederived inhibitor 1; sense oligonucleotides; signal transductioninhibitors; signal transduction modulators; single chain antigen bindingprotein; sizofiran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stem cell inhibitor; stem-cell division inhibitors;stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactiveintestinal peptide antagonist; suradista; suramin; swainsonine;synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide;tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium;telomerase inhibitors; temoporfin; teniposide; tetrachlorodecaoxide;tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietinmimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;titanocene bichloride; topsentin; toremifene; totipotent stem cellfactor; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; vector system, erythrocyte genetherapy; velaresol; veramine; verdins; verteporfin; vinorelbine;vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; andzinostatin stimalamer. Preferred anti-cancer drugs are those that havebeen shown to have treatment benefit in a MPD patient, e.g.,interferon-(hydroxyurea, busulfan, anagrelide, daunorubicin,cincristine, corticosteroid hormones (e.g., prednisone, beclomethasone,cortisone, dexamethasone, fludrocortisone, hydrocortisone,methylprednisolone), kinase inhibitors, topoisomerase inhibitors,famesyl transferase inhibitors, vaccines and antisense nucleotides.

Examples of kinase inhibitors include, but are not limited to, compoundST1571, imatinib mesylate (Kantarjian et al., Clin Cancer Res.8(7):2167-76 (2002)), and those compounds disclosed in U.S. Pat. Nos.6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196,6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769,5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745,5,728,868, 5,648,239, 5,587,459, all of which are incorporated herein byreference. Preferred kinase inhibitors include, but are not limited to,those that directly target the BCR/ABL kinase or other kinases that areinvolved in the MPD pathophysiology, e.g., ST1571, and imatinibmesylate.

Examples of topoisomerase inhibitors include, but are not limited to,camptothecin; irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211(GI 147211); DX-8951f; IST-622; rubitecan; pyrazoloacridine; XR-5000;saintopin; UCE6; UCE1022; TAN-1518A; TAN-1518B; KT6006; KT6528; ED-110;NB-506; ED-110; NB-506; and rebeccamycin; bulgarein; DNA minor groovebinders such as Hoescht dye 33342 and Hoechst dye 33258; nitidine;fagaronine; epiberberine; coralyne; beta-lapachone; BC-4-1; andpharmaceutically acceptable salts, solvates, clathrates, and prodrugsthereof. See, e.g., Rothenberg, M. L., Annals of Oncology8:837-855(1997); and Moreau, P., et al., J. Med. Chem.41:1631-1640(1998). Examples of camptothecin derivatives that can beused in the methods and compositions of this invention are disclosed by,for example, U.S. Pat. Nos. 6,043,367; 6,040,313; 5,932,588; 5,916,896;5,889,017; 5,801,167; 5,674,874; 5,658,920; 5,646,159; 5,633,260;5,604,233; 5,597,829; 5,552,154; 5,541,327; 5,525,731; 5,468,754;5,447,936; 5,446,047; 5,401,747; 5,391,745; 5,364,858; 5,340,817;5,244,903; 5,227,380; 5,225,404; 5,180,722; 5,122,606; 5,122,526;5,106,742; 5,061,800; 5,053,512; 5,049,668; 5,004,758; 4,981,968;4,943,579; 4,939,255; 4,894,456; and 4,604,463, each of which isincorporated herein by reference. Preferred topoisomerase inhibitorsinclude, but are not limited to, DX-8951f, irinotecan, SN-38, andpharmaceutically acceptable salts, solvates, clathrates, and prodrugsthereof.

Examples of farnesyl transferase inhibitor include, but are not limitedto, R115777, BMS-214662, (for review, see Caponigro, Anticancer Drugs13(8):891-897 (2002)), and those disclosed by, for example, U.S. Pat.Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387,6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905,6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501,6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338, 6,228,865,6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786, 6,169,096,6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295,6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935,6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, 6,040,305, all ofwhich are incorporated herein by reference.

In one embodiment of the present invention, the second active agent isan agent used in the gene therapy of MPD. For example, antisenseoligonucleotides can block the encoding instructions of an oncogene sothat it cannot direct the formation of the corresponding oncoproteinthat causes the cell to transform into a malignant cell. Examples ofantisense oligonucleotides include, but are not limited to, thosedisclosed in the U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834,5,734,033, and 5,618,709, all of which are incorporated herein byreference.

In another embodiment of the present invention, the second active agentis a protein, a fusion protein thereof, or a vaccine that secretes theprotein, wherein the protein is IL-2, IL-10, IL-12, IL18, G-CSF, GM-CSF,EPO, or a pharmacologically active mutant or derivative thereof. In somecircumstances apparent to one skilled in the art, G-CSF, GM-CSF and EPOare not preferred. For example, G-CSF, GM-CSF and EPO preferably are notused in methods that do not utilize stem cell transplantation. In apreferred embodiment, the protein is an antibody or an antibody linkedto a chemical toxin or radioactive isotope that targets and killsspecific overproduced cells in a MPD patient. Such antibodies include,but are not limited to, rituximab (Rituxan®), calicheamycin (Mylotarg®),ibritumomab tiuxetan (Zevalin®), and tositumomab (Bexxar®).

In a specific embodiment of the present invention, the second activeagent is a vaccine that can induce antigen-specific anti-malignant cellimmune responses in a MPD patient. A non-limiting example of such avaccine is disclosed in U.S. Pat. No. 6,432,925, which is incorporatedherein by reference.

In yet another embodiment of the present invention, the second activeagent is one that is capable of reversal of multidrug resistance in MPDpatients. The overproduced cells in MPD patients have mechanisms thatmay allow them to escape the damaging effects of chemotherapy. Newagents are being studied to decrease resistance to an importantchemotherapeutic drug used in the treatment of leukemia. Non-limitingexamples of such agents are disclosed in U.S. Pat. No. 6,225,325, whichis incorporated herein by reference.

Other agents that can be used in combination with the present inventioninclude, but are not limited to, those disclosed in U.S. Pat Nos.6,096,300, 6,420,391, 6,326,205, 5,866,332, 6,458,349, 6,420,378,6,399,664, 6,395,771, 6,346,246, 6,333,309, 6,331,642, 6,329,497,6,326,378, 6,313,129, 6,306,393, 6,303,646, 6,265,427, 6,262,053,6,258,779, 6,251,882, 6,231,893, 6,225,323, 6,221,873, 6,218,412,6,204,364, 6,187,287, 6,183,988, 6,183,744, 6,172,112, 6,156,733,6,143,738, 6,127,406, 6,121,320, 6,107,520, 6,107,457, 6,075,015, and6,063,814, all of which are incorporated herein by reference.

4.3 Methods of Treatment and Management

Methods of this invention encompass methods of preventing, treatingand/or managing various types of MPD. As used herein, unless otherwisespecified, the terms “treating” and “preventing” encompass theinhibition or the reduction of the severity or magnitude of one or moresymptoms or laboratory findings associated with MPD. Symptoms associatedwith MPD include, but are not limited to, headache, dizziness, tinnitus,blurred vision, fatigue, night sweat, low-grade fever, generalizedpruritus, epistaxis, blurred vision, splenomegaly, abdominal fullness,thrombosis, increased bleeding, anemia, splenic infarction, severe bonepain, hematopoiesis in the liver, ascites, esophageal varices, liverfailure, respiratory distress, and priapism. Laboratory findingsassociated with MPD include, but are not limited to, clonal expansion ofa multipotent hematopoietic progenitor cell with the overproduction ofone or more of the formed elements of the blood (e.g., elevated redblood cell count, elevated white blood cell count, and/or elevatedplatelet count), presence of Philadelphia chromosome or bcr-abl gene,teardrop poikilocytosis on peripheral blood smear, leukoerythroblasticblood pictuer, giant abnormal platelets, hypercellular bone marrow withreticular or collagen fibrosis, and marked left-shifted myeloid serieswith a low percentage of promyelocytes and blasts. As used herein,unless otherwise specified, the term “treating” refers to theadministration of a composition after the onset of symptoms of MPD,whereas “preventing” refers to the administration prior to the onset ofsymptoms, particularly to patients at risk of MPD. As used herein andunless otherwise indicated, the term “managing” encompasses preventingthe recurrence of MPD in a patient who had suffered from MPD,lengthening the time a patient who had suffered from MPD remains inremission, and/or preventing the occurrence of MPD in patients at riskof suffering from MPD.

The invention encompasses methods of treating or preventing patientswith primary and secondary MPD. It further encompasses methods treatingpatients who have been previously treated for MPD, as well as those whohave not previously been treated for MPD. Because patients with MPD haveheterogenous clinical manifestations and varying clinical outcomes, ithas become apparent that staging the patients according to theirprognosis and approaching therapy depending on the severity and stagemay be necessary. Indeed, the methods and compositions of this inventioncan be used in various stages of treatments for patients with one ormore types of MPD including, but not limited to, polycythemia rubra vera(PRV), primary thromobocythemia (PT), and agnogenic myeloid metaplasia(AMM).

Methods encompassed by this invention comprise administering animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof to a patient (e.g., a human) suffering, or likely to suffer,from MPD. Specific patient populations include the elderly, i.e., ages60 and above as well as those over 35 years of age. Patients withfamilial history of MPD or leukemia are also preferred candidates forpreventive regimens.

In one embodiment of the invention, an immunomodulatory compound of theinvention is administered orally and in a single or divided daily dosesin an amount of from about 0.10 to about 150 mg/day. In a particularembodiment, 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione(Actimid™) is administered in an amount of from about 0.1 to about 5 mgper day. Or about 0.1 to about 1 mg per day, or alternatively from about1 to about 10 mg every other day, or about 5 mg every other day.

3-(4-Amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) can be preferably administered in an amount of from about 2.5to about 25 mg per day. Or about 5 mg to about 10 mg per day, oralternatively from about 5 to about 50 mg every other day, or about 10to about 20 mg every other day. Other dosing regimens for the compoundswill be apparent to the skilled artisan and may involve other dosingschedules known in the art such as, but not limited to, one week ofdaily therapy with the compounds of the invention followed by one weekoff.

4.3.1 Combination Therapy with a Second Active Agent

Particular methods of the invention comprise administering 1) animmunomodulatory compound or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, and 2) asecond active agent or active ingredient. Examples of immunomodulatorycompounds of the invention are disclosed herein (see, e.g., section4.1); and examples of the second active agents are also disclosed herein(see, e.g., section 4.2).

In particular embodiments, one or more immunomodulatory compounds areadministered in combination with the administration of one or moretherapies that are used to treat, manage, or prevent myeloproliferativediseases. A non-limiting example is the use of immunomodulatorycompounds of the invention in combination with the administration of ananti-cancer cocktail regimen, such as, but not limited to, a regimenthat includes cytarabine and an anthracycline (e.g., daunorubicin oridarubicin).

Administration of the immunomodulatory compounds and the second activeagents to a patient can occur simultaneously or sequentially by the sameor different routes of administration. The suitability of a particularroute of administration employed for a particular active agent willdepend on the active agent itself (e.g., whether it can be administeredorally without decomposing prior to entering the blood stream) and thedisease being treated. A preferred route of administration for animmunomodulatory compound is oral. Preferred routes of administrationfor the second active agents or ingredients of the invention are knownto those of ordinary skill in the art. See, e.g., Physicians' DeskReference, 1755-1760 (56^(th) ed., 2002).

In one embodiment, the second active agent is administered intravenouslyor subcutaneously and once or twice daily in an amount of from about 1to about 1000 mg, from about 5 to about 500 mg, from about 10 to about350 mg, or from about 50 to about 200 mg. The specific amount of thesecond active agent will depend on the specific agent used, the type ofMPD being treated or managed, the severity and stage of MPD, and theamount(s) of immunomodulatory compounds of the invention and anyoptional additional active agents concurrently administered to thepatient. In a particular embodiment, the second active agent isinterferon-α, hydroxyurea, anagrelide, arsenic troxide, ST1571, imatinibmesylate, DX-8951f, R115777, vincristine, daunorubicin, prednisone or acombination thereof. Interferon-α is administered in an amount of from 2to 5 million unites subcutaneously three times weekly. Hydroxyurea isadministered in an amount of from about 500 to about 1500 mg/d orally,adjusted to keep platelets <500,000/μL without reducing the neutrophilcount to <2000/μL.

4.3.2 Use with Transplantation Therapy

In still another embodiment, this invention encompasses a method oftreating, preventing and/or managing MPD, which comprises administeringthe immunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof, in conjunction with transplantation therapy. As discussedelsewhere herein, the treatment of MPD is based on the stages andmechanism of the disease. As inevitable leukemic transformation developsin certain stages of MPD, transplantation of peripheral blood stemcells, hematopoietic stem cell preparation or bone marrow may benecessary. The combined use of the immunomodulatory compound of theinvention and transplantation therapy provides a unique and unexpectedsynergism. In particular, an immunomodulatory compound of the inventionexhibits immunomodulatory activity that may provide additive orsynergistic effects when given concurrently with transplantation therapyin patients with MPD. An immunomodulatory compound of the invention canwork in combination with transplantation therapy reducing complicationsassociated with the invasive procedure of transplantation and risk ofrelated Graft Versus Host Disease (GVHD). This invention encompasses amethod of treating, preventing and/or managing MPD which comprisesadministering to a patient (e.g., a human) an immunomodulatory compoundof the invention, or a pharmaceutically acceptable salt, solvate,hydrate, stereoisomer, clathrate, or prodrug thereof, before, during, orafter the transplantation of umbilical cord blood, placental blood,peripheral blood stem cell, hematopoietic stem cell preparation or bonemarrow. Examples of stem cells suitable for use in the methods of theinvention are disclosed in U.S. provisional patent application No.60/372,348, filed Apr. 12, 2002 by R. Hariri et al., the entirety ofwhich is incorporated herein by reference.

4.3.3 Cycling Therapy

In certain embodiments, the prophylactic or therapeutic agents of theinvention are cyclically administered to a patient. Cycling therapyinvolves the administration of an active agent for a period of time,followed by a rest for a period of time, and repeating this sequentialadministration. Cycling therapy can reduce the development of resistanceto one or more of the therapies, avoid or reduce the side effects of oneof the therapies, and/or improves the efficacy of the treatment.

Consequently, in one specific embodiment of the invention, animmunomodulatory compound of the invention is administered daily in asingle or divided doses in a four to six week cycle with a rest periodof about a week or two weeks. The invention further allows thefrequency, number, and length of dosing cycles to be increased. Thus,another specific embodiment of the invention encompasses theadministration of an immunomodulatory compound of the invention for morecycles than are typical when it is administered alone. In yet anotherspecific embodiment of the invention, an immunomodulatory compound ofthe invention is administered for a greater number of cycles that wouldtypically cause dose-limiting toxicity in a patient to whom a secondactive ingredient is not also being administered.

In one embodiment, an immunomodulatory compound of the invention isadministered daily and continuously for 3 or 4 weeks at a dose of fromabout 0.1 to about 150 mg/d followed by a break of 1 or 2 weeks.Actimid™ is preferably administered daily and continuously at an initialdose of 0.1 to 5 mg/d with dose escalation (every week) by 1 to 10 mg/dto a maximum dose of 50 mg/d for as long as therapy is tolerated. In aparticular embodiment, Revimid™ is administered in an amount of about 5,10, or 25 mg/day, preferably in an amount of about 10 mg/day for threeto four weeks, followed by one week or two weeks of rest in a four orsix week cycle.

In one embodiment of the invention, an immunomodulatory compound of theinvention and a second active ingredient are administered orally, withadministration of an immunomodulatory compound of the inventionoccurring 30 to 60 minutes prior to a second active ingredient, during acycle of 4 to 6 weeks. In another embodiment of the invention, thecombination of an immunomodulatory compound of the invention and asecond active ingredient is administered by intravenous infusion overabout 90 minutes every cycle. In a specific embodiment, one cyclecomprises the administration of from about 10 to about 25 mg/day ofRevimid™ and from about 50 to about 200 mg/m²/day of a second activeingredient daily for 3 to 4 weeks and then one or two weeks of rest. Inanother specific embodiment, each cycle comprises the administration offrom about 5 to about 10 mg/day of Actimid™ and from about 50 to about200 mg/m² /day of a second active ingredient for 3 to 4 weeks followedby one or two weeks of rest. Typically, the number of cycles duringwhich the combinatorial treatment is administered to a patient will befrom about 1 to about 24 cycles, more typically from about 2 to about 16cycles, and even more typically from about 4 to about 8 cycles.

4.4 Pharmaceutical Compositions and Single Unit Dosage Forms

Pharmaceutical compositions can be used in the preparation ofindividual, single unit dosage forms. Pharmaceutical compositions anddosage forms of the invention comprise an immunomodulatory compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof. Pharmaceutical compositionsand dosage forms of the invention can further comprise one or moreexcipients.

Pharmaceutical compositions and dosage forms of the invention can alsocomprise one or more additional active ingredients. Consequently,pharmaceutical compositions and dosage forms of the invention comprisethe active ingredients disclosed herein (e.g., an immunomodulatorycompound of the invention, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, and asecond active ingredient). Examples of optional additional activeingredients are disclosed herein (see, e.g., section 5.2).

Single unit dosage forms of the invention are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), or parenteral(e.g., subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), transdermal or transcutaneous administration to apatent. Examples of dosage forms include, but are not limited to:tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; powders;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use. For example, a dosage form usedin the acute treatment of a disease may contain larger amounts of one ormore of the active ingredients it comprises than a dosage form used inthe chronic treatment of the same disease. Similarly, a parenteraldosage form may contain smaller amounts of one or more of the activeingredients it comprises than an oral dosage form used to treat the samedisease. These and other ways in which specific dosage forms encompassedby this invention will vary from one another will be readily apparent tothose skilled in the art. See, e.g., Remington's PharmaceuticalSciences, 18th ed., Mack Publishing, Easton Pa. (1990).

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsare provided herein. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patient.For example, oral dosage forms such as tablets may contain excipientsnot suited for use in parenteral dosage forms. The suitability of aparticular excipient may also depend on the specific active ingredientsin the dosage form. For example, the decomposition of some activeingredients may be accelerated by some excipients such as lactose, orwhen exposed to water. Active ingredients that comprise primary orsecondary amines are particularly susceptible to such accelerateddecomposition. Consequently, this invention encompasses pharmaceuticalcompositions and dosage forms that contain little, if any, lactose othermono- or di-saccharides. As used herein, the term “lactose-free” meansthat the amount of lactose present, if any, is insufficient tosubstantially increase the degradation rate of an active ingredient.

Lactose-free compositions of the invention can comprise excipients thatare well known in the art and are listed, for example, in the U.S.Pharmacopeia (USP) 25-NF20 (2002). In general, lactose-free compositionscomprise active ingredients, a binder/filler, and a lubricant inpharmaceutically compatible and pharmaceutically acceptable amounts.Preferred lactose-free dosage forms comprise active ingredients,microcrystalline cellulose, pre-gelatinized starch, and magnesiumstearate.

This invention further encompasses anhydrous pharmaceutical compositionsand dosage forms comprising active ingredients, since water canfacilitate the degradation of some compounds. For example, the additionof water (e.g., 5%) is widely accepted in the pharmaceutical arts as ameans of simulating long-term storage in order to determinecharacteristics such as shelf-life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect,water and heat accelerate the decomposition of some compounds. Thus, theeffect of water on a formulation can be of great significance sincemoisture and/or humidity are commonly encountered during manufacture,handling, packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms of the inventioncan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichan active ingredient will decompose. Such compounds, which are referredto herein as “stabilizers,” include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. However, typical dosage forms of the invention comprise animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof in an amount of from about 0.10 to about 150 mg. Typical dosageforms comprise an immunomodulatory compound of the invention, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof in an amount of about 0.1, 1, 2, 5, 7.5,10, 12.5, 15, 17.5, 20, 25, 50, 100, 150 or 200 mg. In a particularembodiment, a preferred dosage form comprises4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (Actimid™) inan amount of about 1, 2, 5, 10, 25 or 50 mg. In a specific embodiment, apreferred dosage form comprises3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) in an amount of about 5, 10, 25 or 50 mg. Typical dosageforms comprise the second active ingredient in an amount of 1 to about1000 mg, from about 5 to about 500 mg, from about 10 to about 350 mg, orfrom about 50 to about 200 mg. Of course, the specific amount of thesecond active ingredient will depend on the specific agent used, thetype of MPD being treated or managed, and the amount(s) ofimmunomodulatory compounds of the invention, and any optional additionalactive agents concurrently administered to the patient.

4.4.1 Oral Dosage Forms

Pharmaceutical compositions of the invention that are suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

Typical oral dosage forms of the invention are prepared by combining theactive ingredients in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of theinvention include, but are not limited to, binders, fillers,disintegrants, and lubricants. Binders suitable for use inpharmaceutical compositions and dosage forms include, but are notlimited to, corn starch, potato starch, or other starches, gelatin,natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropylmethyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystallinecellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, PA), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the invention istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Disintegrants are used in the compositions of the invention to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may disintegrate in storage,while those that contain too little may not disintegrate at a desiredrate or under the desired conditions. Thus, a sufficient amount ofdisintegrant that is neither too much nor too little to detrimentallyalter the release of the active ingredients should be used to form solidoral dosage forms of the invention. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Typical pharmaceutical compositionscomprise from about 0.5 to about 15 weight percent of disintegrant,preferably from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, agar-agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, other starches, clays, other algins, other celluloses, gums, andmixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

A preferred solid oral dosage form of the invention comprises animmunomodulatory compound of the invention, anhydrous lactose,microcrystalline cellulose, polyvinylpyrrolidone, stearic acid,colloidal anhydrous silica, and gelatin.

4.4.2 Delayed Release Dosage Forms

Active ingredients of the invention can be administered by controlledrelease means or by delivery devices that are well known to those ofordinary skill in the art. Examples include, but are not limited to,those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548,5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which isincorporated herein by reference. Such dosage forms can be used toprovide slow or controlled-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the active ingredients of the invention. The invention thusencompasses single unit dosage forms suitable for oral administrationsuch as, but not limited to, tablets, capsules, gelcaps, and capletsthat are adapted for controlled-release.

All controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood levels of the drug,and can thus affect the occurrence of side (e.g., adverse) effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled-release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

4.4.3 Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are preferably sterile orcapable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms of the invention. For example, cyclodextrin andits derivatives can be used to increase the solubility of animmunomodulatory compound of the invention, and its derivatives. See,e.g., U.S. Pat. No. 5,134,127, which is incorporated herein byreference.

4.4.4 Topical and Mucosal Dosage Forms

Topical and mucosal dosage forms of the invention include, but are notlimited to, sprays, aerosols, solutions, emulsions, suspensions, orother forms known to one of skill in the art. See, e.g., Remington'sPharmaceutical Sciences, 16^(th) and 18^(th) eds., Mack Publishing,Easton Pa. (1980 & 1990); and Introduction to Pharmaceutical DosageForms, 4th ed., Lea & Febiger, Philadelphia (1985). Dosage formssuitable for treating mucosal tissues within the oral cavity can beformulated as mouthwashes or as oral gels.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide topical and mucosal dosage forms encompassedby this invention are well known to those skilled in the pharmaceuticalarts, and depend on the particular tissue to which a givenpharmaceutical composition or dosage form will be applied. With thatfact in mind, typical excipients include, but are not limited to, water,acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol,isopropyl myristate, isopropyl palmitate, mineral oil, and mixturesthereof to form solutions, emulsions or gels, which are non-toxic andpharmaceutically acceptable. Moisturizers or humectants can also beadded to pharmaceutical compositions and dosage forms if desired.Examples of such additional ingredients are well known in the art. See,e.g., Remington's Pharmaceutical Sciences, 16^(th) and 18^(th) eds.,Mack Publishing, Easton Pa. (1980 & 1990).

The pH of a pharmaceutical composition or dosage form may also beadjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

4.4.5 Kits

Typically, active ingredients of the invention are preferably notadministered to a patient at the same time or by the same route ofadministration. This invention therefore encompasses kits which, whenused by the medical practitioner, can simplify the administration ofappropriate amounts of active ingredients to a patient.

A typical kit of the invention comprises a dosage form of animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, prodrug, or clathratethereof. Kits encompassed by this invention can further compriseadditional active ingredients such as, but not limited to, interferon-α,hydroxyurea, anagrelide, arsenic troxide, ST1571, imatinib mesylate,DX-8951f, R115777, vincristine, daunorubicin, prednisone, or apharmacologically active mutant or derivative thereof, or a combinationthereof. Examples of the additional active ingredients include, but arenot limited to, those disclosed herein (see, e.g., section 4.2).

Kits of the invention can further comprise devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, drip bags, patches, and inhalers.

Kits of the invention can further comprise cells or blood fortransplantation as well as pharmaceutically acceptable vehicles that canbe used to administer one or more active ingredients. For example, if anactive ingredient is provided in a solid form that must be reconstitutedfor parenteral administration, the kit can comprise a sealed containerof a suitable vehicle in which the active ingredient can be dissolved toform a particulate-free sterile solution that is suitable for parenteraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

5. EXAMPLES

The following studies are intended to further illustrate the inventionwithout limiting its scope.

5.1 Pharmacology and Toxicology Studies

A series of non-clinical pharmacology and toxicology studies have beenperformed to support the clinical evaluation of an immunomodulatorycompound of the invention in human subjects. These studies wereperformed in accordance with internationally recognized guidelines forstudy design and in compliance with the requirements of Good LaboratoryPractice (GLP), unless otherwise noted.

The pharmacological properties of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione,including activity comparisons with thalidomide, have been characterizedin vitro studies. Studies examined the effects of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione orthalidomide on the production of various cytokines. In all studies,3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione was atleast 50 times more potent than thalidomide. In addition, a safetypharmacology study of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione hasbeen conducted in dogs and the effects of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione on ECGparameters were examined further as part of three repeat-dose toxicitystudies in primates. The results of these studies are described below.

5.2 Modulation of Cytokine Production

Inhibition of TNF-α production following LPS-stimulation of human PBMCand human whole blood by3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione orthalidomide was investigated in vitro (Muller et al., Bioorg. Med. Chem.Lett. 9:1625-1630, 1999). The IC₅₀'s of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine2,6-dione forinhibiting production of TNF-α following LPS-stimulation of PBMC andhuman whole blood were ˜100 nM (25.9 ng/mL) and ˜480 nM (103.6 ng/mL),respectively. Thalidomide, in contrast, had an IC₅₀ of ˜194 μM (50.2μg/mL) for inhibiting production of TNF-α following LPS-stimulation ofPBMC.

5.3 Toxicology Studies

The effects of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione oncardiovascular and respiratory function were investigated inanesthetized dogs. Two groups of Beagle dogs (2/sex/group) were used.One group received three doses of vehicle only and the other receivedthree ascending doses of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(2, 10,and 20 mg/kg). In all cases, doses of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione orvehicle were successively administered via infusion through the jugularvein separated by intervals of at least 30 minutes.

No animals died in this study. The cardiovascular and respiratorychanges induced by3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione wereminimal at all doses when compared to the vehicle control group. Theonly statistically significant difference between the vehicle andtreatment groups was a small increase in arterial blood pressure (from94 mmHg to 101 mmHg) following administration of the low dose of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Thiseffect lasted approximately 15 minutes and was not seen at higher doses.Deviations in femoral blood flow, respiratory parameters, and Qtcinterval were common to both the control and treated groups and were notconsidered treatment-related.

All patents cited herein are incorporated by reference in theirentireties. Embodiments of the invention described herein are only asampling of the scope of the invention. The full scope of the inventionis better understood with reference to the attached claims.

1. A method of treating a myeloproliferative disease, which comprisesadministering to a patient having a myeloproliferative disease atherapeutically effective amount of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione, or apharmaceutically acceptable salt thereof, wherein the myeloproliferativedisease is selected from the group consisting of polycythemia rubravera, primary thrombocythemia, and agnogenic myeloid metaplasia, andwherein the therapeutically effective amount is from about 1 mg to about50 mg per day.
 2. A method of treating a myeloproliferative disease,which comprises administering to a patient having a myeloproliferativedisease from about 5 mg to about 50 mg per day of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione, or apharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of at least one second active agent, wherein themyeloproliferative disease is selected from the group consisting ofpolycythemia rubra vera, primary thrombocythemia, and agnogenic myeloidmetaplasia.
 3. The method of claim 1 or 2, wherein the patient isrefractory to a myeloproliferative disease treatment comprisingthalidomide.
 4. The method of claim 2, wherein the second active agentis a corticosteroid, platelet inhibitor, anticoagulant, thrombolyticagent, antifibrosis agent, all-trans retinoic acid, topoisomeraseinhibitor, farnesyl transferase inhibitor, myelosuppressive agent oranti-cancer agent.
 5. The method of claim 4, wherein the second activeagent is interferon-α, hydroxyurea, anagrelide, busulfan, arsenictrioxide, imatinib mesylate, exatecan mesylate, tipifarnib, vincristine,daunorubicin, prednisone, or a combination thereof.
 6. The method ofclaim 1 or 2, wherein the myeloproliferative disease is primary orsecondary.
 7. A method of treating a myeloproliferative disease, whichcomprises administering to a patient having a myeloproliferative diseasea therapeutically effective amount of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione, or apharmaceutically acceptable salt thereof, before, during or aftertransplanting umbilical cord blood, placental blood, peripheral bloodstem cell, hematopoietic stem cell preparation or bone marrow into thepatient wherein the myeloproliferative disease is selected from thegroup consisting of polycythemia rubra vera, primary thrombocythemia,and agnogenic myeloid metaplasia, and wherein the therapeuticallyeffective amount is from about 5 mg to about 50 mg per day.
 8. Themethod of claim 1, 2, or 7, wherein the 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione is administered orally. 9.The method of claim 8, wherein the 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione is administered in the formof a capsule or tablet.
 10. The method of claim 1, 2, or 7, wherein the3-(4-amino-1-oxo-1,3- dihydro-isoindol-2-yl)-piperidine-2,6-dione isadministered in an amount of about 5 mg, 10 mg, 25 mg or 50 mg per day.11. The method of claim 1, 2, or 7, wherein the 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione is administered in an amountof from about 5 mg to about 25 mg per day.
 12. The method of claim 1, 2,or 7, wherein the 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione is administered in an amountof about 25 mg per day.
 13. The method of claim 1, 2, or 7, wherein the3-(4-amino-1-oxo-1,3- dihydro-isoindol-2-yl)-piperidine-2,6-dione is apharmaceutically acceptable salt.
 14. A method of treating agnogenicmyeloid metaplasia, which comprises administering to a patient havingagnogenic myeloid metaplasia from about 5 mg to about 50 mg per day of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione and atherapeutically effective amount of rituximab.
 15. A method of treatingagnogenic myeloid metaplasia, which comprises administering to a patienthaving agnogenic myeloid metaplasia from about 5 mg to about 50 mg perday of 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dioneand a therapeutically effective amount of fludarabine.
 16. The method ofclaim 1, 2, or 7, wherein the compound is 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione, having the formula:

as a free base.